Mitomycin c

Manufactured by Thermo Fisher Scientific

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Mitomycin C is a laboratory reagent used in various biological and pharmaceutical applications. It is an antibiotic and antitumor agent derived from the bacterium Streptomyces caespitosus. Mitomycin C functions as a DNA cross-linking agent, inhibiting DNA synthesis and cell division.

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Mitomycin (2 citations)

70 protocols using mitomycin c

Evaluating T-cell Response to Tumour Cells

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Tumour tissues were harvested 7 days after the last exosome injection and fixed with 4% paraformaldehyde. The CD8+ or CD4+ lymphocytes in tumours were stained, as previously described (Du et al, 2006 (link)). To evaluate the T-cell response against Hepa 1–6 tumour cells in vitro, spleens were harvested from Hepa 1–6 inoculated mice in each group 7 days after last exosome injection. Splenocytes were isolated and co-cultured with Hepa 1–6 cells (ratio of 10 : 1, respectively) and treated with mitomycin C (Fisher Scientific) or MC38.
mitomycin C treatment was completed as follows: Hepa 1–6 and MC38 cells were treated with mitomycin C (50 μg ml⁻¹) in media for 1 h, and then the culture medium was removed and cells were washed with PBS three times. During the co-culturing of splenocytes and tumour cells, 30 U ml⁻¹ recombinant human IL-2 (R&D) was added to the media for 5 days. The cells were harvested and flow cytometry was used to analyse intracellular IFNγ and granzyme B in CD8+ cells. Cytofix/Cytoperm Fixation/Permeabilisation kit (BD Bioscience) was used to prepare cells for surface and intracellular flow cytometry staining, according to the protocol suggested by the manufacturer.

Yang M.Q., Du Q., Varley P.R., Goswami J., Liang Z., Wang R., Li H., Stolz D.B, & Geller D.A. (2017). Interferon regulatory factor 1 priming of tumour-derived exosomes enhances the antitumour immune response. British Journal of Cancer, 118(1), 62-71.

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Vibriophage Induction by Mitomycin C

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Induction of vibriophage by mitomycin C was performed according to the methodology mentioned by Castillo et al. [48 (link)], with slight modification. In brief, preserved Vibrio species in LB agar stab were aseptically transferred with a sterile inoculum loop to 10 mL of autoclaved LB media and incubated at 37 °C overnight at 200 rpm in an incubator shaker (ThermoFisher Scientific, Waltham, MA, USA). The overnight grown culture was sub-cultured in LB medium and incubated again at approximately 37 °C for 2 h at 200 rpm to reach the Vibrio culture optical density (OD₅₅₀) of ≥0.2 at 550 nm. Phage induction was initiated by the addition of mitomycin C (at a final concentration of 0.5 µg/mL) (ThermoFisher Scientific, MA, USA) in grown culture (MMC+), except for the control (MMC−) without mitomycin C. Then, the optical density (OD₆₀₀) of samples was measured at a time interval of 20 min for 8 h.

Pandey R., Sharma S, & Sinha K.K. (2023). Evidence of Antibiotic Resistance and Virulence Factors in Environmental Isolates of Vibrio Species. Antibiotics, 12(6), 1062.

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Induction of Murine Regulatory T Cells In Vitro

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Naïve CD4+ T splenocytes were sorted as GFP⁻CD4⁺CD44^lowCD62L^high from Foxp3-gfp reporter mice (conjugated mAbs from BioLegend) on a Moflo sorter. Two different protocols were then used: in the first, T cells were activated with anti-CD3/CD28–coated beads (Thermo Fisher) at a concentration of two cells per bead in the presence of 2 U/ml of human rIL-2 and 10 ng/ml rTGF-β (Peprotech) in RPMI with 10% FCS (Chen et al., 2003 (link)). In the second, T cells were activated with 1μg/ml soluble anti-CD3ε (Thermo) presented by T-depleted Mitomycin C (Sigma)-inactivated splenocytes (1:3 ratio) in the presence of 2 U/ml rhIL-2 and 10 ng/ml rTGF-β in RPMI with 10% FCS (Wheaton et al., 2017 (link)). To prepare these presenting cells, spleen suspension from congenic B6.CD45.1 mice were depleted of T cells by incubation with Biotin-conjugated anti-CD3ε (Biolegend) and anti-TCRβ in Dynal buffer (1mg/ml BSA and 2 mM EDTA in PBS) for 20min on ice, washed and incubated with Dynabeads BiotinBinder (150ul per spleen, Thermo) for 30min at 4°C with rotation. T cells were removed using DynaMag magnet (Thermo), and the remaining cells blocked with 0.5mg/ml Mitomycin C at 37°C for 2hs. After 3 days in culture, cells were analyzed by flow cytometry.

Yan Y., Ramanan D., Rozenberg M., McGovern K., Rastelli D., Vijaykumar B., Yaghi O., Voisin T., Mosaheb M., Chiu I., Itzkovitz S., Rao M., Mathis D, & Benoist C. (2021). Interleukin 6 produced by enteric neurons regulates the number and phenotype of microbe-responsive regulatory T cells in the gut. Immunity, 54(3), 499-513.e5.

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Differentiation of human embryonic stem cells into excitatory postsynaptic cells

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hEPSC were cultured and maintained as described^[⁵^] with minor modifications. Briefly, the cells were cultured on mitomycin‐C (Thermo Fisher Scientific) inactivated STO feeder cells, which were seeded on 0.1% gelatin (Sigma Aldrich)‐coated wells at a density of 0.075 × 10⁶ cells/cm² at least 2 days prior to hEPSC seeding. The hEPSC medium: DMEM/F12, 1× L‐glutamine, 1× penicillin–streptomycin, 1× NEAA, 0.1 µm 2‐mercaptoethanol, 1× N2 supplement, 1× B27 supplement (Thermo Fisher Scientific), and 65 µg mL⁻¹ L‐ascorbic acid (Sigma Aldrich) supplemented with 2.5 µm XAV939 (Sigma Aldrich), 0.15 µm A419259 (Tocris); 1.0 µm CHIR99021 (Stemgent), 0.25 µm SB590885 (R&D), and 10 ng mL⁻¹ recombinant human LIF (PeproTech). 20% Knock‐out serum replacement (KOSR; Thermo Fisher Scientific) and 10 µm Y‐27632 (Stemcell Technologies) were supplemented to the medium on the day of hEPSC seeding and hEPSC were passaged every 3–4 days.

Chen A.C., Lee Y.L., Ruan H., Huang W., Fong S.W., Tian S., Lee K.C., Wu G.M., Tan Y., Wong T.C., Wu J., Zhang W., Cao D., Chow J.F., Liu P, & Yeung W.S. (2023). Expanded Potential Stem Cells from Human Embryos Have an Open Chromatin Configuration with Enhanced Trophoblast Differentiation Ability. Advanced Science, 10(11), 2204797.

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Induction of Quorum Sensing Molecules

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Mitomycin C was purchased from two different vendors: Thermo Fisher Scientific (Waltham, MA, USA) and ApexBio (Houston, TX, USA); irrespective of source, Mitomycin C was added to induced samples at a final concentration of 0.5 µg mL⁻¹. SedgeHammer (Gowan Company, Yuma, AZ, USA) herbicide was prepared by dissolving 0.5 g of SedgeHammer in 10 mL of sterile deionized water. SedgeHammer was added to induced samples at a final concentration of 0.05 µg mL⁻¹ (37 ). An equimolar mixture of acyl-homoserine lactones (N-Hexanoyl-L-homoserine lactone and N-Tetradecanoyl-DL-homoserine lactone, Sigma-Aldrich, St. Louis, MO, USA) was used, and the AHL mixture was added to induced samples to achieve a final concentration of 1 µM ((13 (link)).

Jacoby M.L., Hogg G.D., Assaad M.R, & Williamson K.E. (2023). Seasonal trends in lysogeny in an Appalachian oak-hickory forest soil. Applied and Environmental Microbiology, 90(1), e01408-23.

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Murine 3T3 Fibroblast Cryopreservation

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Murine 3T3 fibroblasts were cultured in DMEM (Gibco, USA) enriched with 10% of Bovine calf serum defined/supplemented (BCS, HyClone, USA) medium for 5 days, at an initial density of 1.5 × 10⁶ cells, in an incubator at 37 °C and in a humidified atmosphere containing 5% of CO₂, with a medium change every two days. When 3T3 covered 70–80% of the flask area, they were treated with a solution of 0.5 mg/ml of Mitomycin C (Sigma, USA) and Dulbecco's phosphate-buffered saline enriched with calcium and magnesium (PBS+, ThermoFisher, USA) solution for two hours in a 37 °C/5% CO₂ incubator. Mitomycin C-treated 3T3 were washed with phosphate buffered saline (PBS, ThermoFisher, USA) and harvested at passage 13 using 0.05% Trypsin EDTA (1×) (Gibco, USA), resuspended in DMSO (Sigma, USA) 7.5% diluted in DMEM + 15% of BCS and homogenously distributed in cryopreserved vial at 1 × 10⁷ cells per vial and stored in liquid nitrogen for future use.

Madiedo-Podvrsan S., Belaïdi J.P., Desbouis S., Simonetti L., Ben-Khalifa Y., Collin-Djangone C., Soeur J, & Rielland M. (2021). Utilization of patterned bioprinting for heterogeneous and physiologically representative reconstructed epidermal skin models. Scientific Reports, 11, 6217.

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Targeted Silencing of RAD51 DNA Repair

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Oligonucleotides were purchased from Sigma or IDT. ON-TARGET plus siRNA to RAD51 from Dharmacon. Mission siRNA and universal negative control #1 was from Sigma-Aldric. Mitomycin C (MMC) was from Thermo Fisher. Restriction enzymes from New England Biolabs. All other chemicals were analytical grade or better.

McKinzey D.R., Gomathinayagam S., Griffin W.C., Klinzing K.N., Jeffries E.P., Rajkovic A, & Trakselis M.A. (2021). Motifs of the C-terminal domain of MCM9 direct localization to sites of mitomycin-C damage for RAD51 recruitment. The Journal of Biological Chemistry, 296, 100355.

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T Cell Proliferation Assay

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B6 CD4⁺ T cells were purified using a negative selection kit from the spleen, as described above. T cells were labeled with CellTrace violet proliferation dye (Invitrogen) and co-cultured in a 1:1 ratio with BALB/c splenocytes treated with mitomycin-C (25 μg/ml) (Thermo-Fischer). Cell proliferation was analyzed on day 7 by flow cytometry.

Aggarwal N., Deerhake M.E., DiPalma D., Shahi S.K., Gaggioli M.R., Mangalam A.K, & Shinohara M.L. (2021). Secreted osteopontin from CD4+ T cells limits acute graft-versus-host disease. Cell reports, 37(13), 110170.

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Cell Invasion Assay Using Matrigel

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Cells were pre-treated with 10ug/mL mitomycin C (Thermo Fisher Scientific) for two hours prior to assay. A total of 1x10⁵ cells were suspended using FBS-free culture medium and seeded onto the Matrigel^®-pre-coated (Sigma-Aldrich, St. Louis, MO, USA) upper chamber (BD Biosciences, Franklin Lakes, New Jersey, USA). Subsequently, 500 μl of culture medium containing 10% FBS was added into the lower chamber. After overnight incubation, non-invasive cells were detached using a cotton swab, while invaded cells in the lower chamber were fixed using 4% paraformaldehyde and stained by 0.5% crystal violet. The numbers of invasive cells were counted in five randomly selected fields using an inverted microscope (magnificationx200, Olympus Corporation, Japan).

Wang Z, & Liu C. (2021). Upregulated hsa_circRNA_100269 inhibits the growth and metastasis of gastric cancer through inactivating PI3K/Akt axis. PLoS ONE, 16(4), e0250603.

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Wound Healing Assay in Liver Cancer Cells

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Transfected Huh-7 and Hep3B2.1–7 cells (1×10⁵) were seeded into a 6-well plate, cultured for 24 h, and then treated with 10 µg/ml Mitomycin C (Thermo Fisher Scientific, Inc.) for 2 h to halt proliferation. A wound was created through the monolayer using a 100-µl pipette tip, and the cells were cultured in serum-free medium. Wound-closure images were captured under a microscope (DMI3000 B; Leica Microsystems, Inc.; magnification, ×100) at 0 and 24 h. The wound healing rate was calculated as follows: (0 h width of scratch–24 h width of scratch)/0 h width of scratch ×100%.

Ding Q., Lin D., Zhou Y., Li F., Lai J., Duan J., Chen J, & Jiang C. (2021). Downregulation of amine oxidase copper containing 1 inhibits tumor progression by suppressing IL-6/JAK/STAT3 pathway activation in hepatocellular carcinoma. Oncology Letters, 22(6), 857.

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